Acidophilic Enzymes
a technology of acidophilic enzymes and enzymes, applied in the field of acidophilic enzymes, can solve problems such as dna damage, and achieve the effect of good stability and good stability of each enzym
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example 2
Expression of Acidophilic Ligase LigFA in E. coli
[0048]DNA fragments encoding LigFA were excised from plasmids according to Example 1 and isolated by gel electrophoresis, then ligated into an expression vector (e.g. PET-3a by Novagen), pre-digested with the same endonucleases and dephosphorylated. After transformation of an E. coli expression host (E. coli BL21), transformants were used for heterologous expression of LigFA using LB-medium containing 100 μM FeCl2 and appropriate antibiotics. For induction of expression, 2 mM IPTG were added to overnight cultures, diluted ten-fold with fresh and pre-warmed LB-medium containing 100 μM FeCl2. For isolation of LigFA, an induction period of about two hours was found to be sufficient. Cells were harvested by centrifugation and resuspended in 10 mM sodium citrate buffer, pH 3.0, containing 100 μM FeCl2 and protease inhibitors as well as DNase. After sonication and separation from cell debris by centrifugation (10,000×g, 30 minutes, 4° C.) ...
example 3
DNA Ligation Using Acidophilic DNA Ligase (LigFA)
[0049]For DNA ligation, double-stranded DNA fragments, each comprising a complementary 3′ overhang which were phosphorylated in 5′ can be ligated in a total volume of 20 μL ligation buffer (100 mM Na-citrate, pH 3.0, 10 μM Fe2+, 0.01-0.1 mM ATP, optionally 0.5 mM dithiothreitol), 0.1-5 micrograms DNA and 1 to 20 nmoles ligase. The reaction conditions are 40° C. for 5 minutes to 2 hours, the reaction can be stopped by the addition of stop buffer 98% (vol / vol) formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol, 0.2% SDS), followed by heating to 95° C. for 5 minutes or, alternatively, by adding stop / loading buffer (30% sucrose, 150 mM EDTA, 0.15% SDS, 0.03% bromophenol blue), and heating to 90° C. for 2 minutes.
[0050]When ligating two short oligonucleotides (35 bases and 25 bases) complementary to a 70 base oligonucleotide, using the same buffer at varying pH values, in a reaction volume of 20 μL, using 20 nM ligFA at 40°...
example 4
Religation Activity of LigFA of Topoisomerase 2 Induced DNA Breaks
[0054]For a comparison of the religation activities of acidic LigFA to T4 ligase on DNA containing breaks caused by topoisomerase 2 (TOP 2), ligation activities were determined in vitro at various pH values. The results are shown in the non-denaturing agarose gel of FIG. 9.
[0055]DNA was incubated with TOP 2 to induce DNA damage. At pH 5.0, TOP 2 caused DNA breaks (lane 2) but not at pH 3.0 (lane 3). Using TOP 2 fragmented DNA (pH 5.0), religation activity of LigFA was clearly demonstrated using acidic reaction conditions (pH 4.0 to 2.0, lanes 7 to 10, respectively) by generation of larger DNA fragments. The comparative T4 ligase did not catalyse re-ligation of TOP 2 fragmented DNA at acidic pH values (lanes 13 to 17), but was active at pH above 7.5 (lane 18). However, at pH 7.5 TOP 2 did not show damaging activity (lane 19).
[0056]This example shows that acidic DNA ligases according to the invention can re-ligate TOP 2...
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